The Arabidopsis immune receptor EFR increases resistance to the bacterial pathogens Xanthomonas and Xylella in transgenic sweet orange

Citrus agribusiness faces major economic losses due to bacterial diseases (Caserta et al., 2020). Citrus canker (CC) and citrus variegated chlorosis (CVC) caused by Xanthomonas citri subsp. citri (Xcc) and Xylella fastidiosa subsp. pauca (Xfp), respectively, are important threats in commercial citrus orchards. All sweet orange (Citrus sinensis) commercial varieties are susceptible to both pathogens, and no natural resistance has been found so far. Plant cell-surface receptors recognize pathogen (or microbe)-associated molecular patterns (PAMPs/MAMPs) to activate pattern-triggered immunity (PTI). The well-studied Arabidopsis thaliana ELONGATION FACTOR-TU RECEPTOR (EFR) recognizes the conserved bacterial PAMP EF-Tu and derived elf peptides (Boutrot and Zipfel, 2017). Interfamily transfer of EFR has been shown to increase anti-bacterial disease resistance in several crops. The manipulation of PTI-related genetic traits has the potential to create more durable resistance assuring a sustainable productivity (Boutrot and Zipfel, 2017). EF-Tu is present in the biofilm of both bacteria (Silva et al., 2011; Zimaro et al., 2013) and the outer membrane vesicles (OMVs) released by X. fastidiosa (Feitosa-Junior et al., 2019). We hypothesized that EFR gene transfer is a powerful strategy to increase Citrus broad-spectrum resistance against CC and CVC. We first used the genetic models Arabidopsis and Nicotiana tabacum to address whether EFR can recognize these bacteria. Arabidopsis Col-0 (wild-type, WT) produced reactive oxygen species (ROS) when challenged with living Xylella fastidiosa subsp. fastidiosa (Xff), which was markedly reduced in efr-1 mutants (Figure 1a). Similarly, Arabidopsis seedling growth was strongly repressed in the presence of Xff OMVs (Figure 1b). No growth repression was observed in efr-1 or bak1-5, a mutant affected in the EFR co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) (Schwessinger et al., 2011). We then examined whether EFR modulates Xff infection in Arabidopsis (Pereira et al., 2019). Compared to Col-0, efr-1 supported higher bacterial loads, quantified as HL5/HL6 abundance (Figure 1c). These observations suggest the effective perception of Xff EF-Tu by EFR, showing that it is sufficient to restrict Xff colonization in Arabidopsis. We next investigated whether Xfp- and Xcc-derived elf peptides activate EFR-dependent immune responses. We tested ROS production in Col-0 and transgenic tobacco expressing EFR, both showing ROS production quickly after elf18Ec (E. coli) exposure. Although there are sequence differences between the elf peptides (Figure 1g), ROS was similarly produced in Col-0 after elf18Xcc or elf26Xfp treatment, albeit to a lower extent in the later case (Figure 1d). A similar pattern was observed for transgenic tobacco expressing EFR (Figure 1e). Together, these findings indicate that EFR recognize EF-Tu from citrus phytobacteria, indicating that its transfer to sweet orange might be a good strategy to confer broad-spectrum recognition. To test whether EFR is functional in citrus, nine independent transgenic lines of Valencia sweet orange (V1 to V9) expressing EFR were obtained. Transgene integration and expression were confirmed through histochemical GUS assay and RT-qPCR, respectively (Figure 1f). Each line had a single transgene copy. Seedlings were grafted in Rangpur lime rootstocks, showing normal development. Transgenic leaf discs challenged with elf18Ec, elf18Xcc or elf26Xfp showed variable levels of ROS production depending on the peptide (Figure 1h). Treatment with elf18Ec and elf18Xcc produced similar results, although slightly delayed for elf18Xcc. The lines V4 and V5 showed responsiveness to elf26Xfp but at relatively low level (Figure 1h). The elf-induced ROS production in EFR transgenic citrus indicates functional conservation of the required intracellular signalling components in sweet orange. Since V4 and V5 responded to the three peptides, they were further tested for the activation of mitogen-activated kinases (MAPK) and expression of defence genes. MAPK phosphorylation was detected in both lines compared to the mock treatment 30 and 45 min after elf treatment, with stronger signal after 45 min (Figure 1i). Interestingly, constitutive activation was observed in the V5 line; yet the signal further increased after peptide treatment. The citrus defence genes SGT1, EDS1, WRKY23 and NPR2 (Shi et al., 2015) revealed that all genes were induced 3 h after peptide treatment at different extents in the transgenic lines compared to respective mock samples (Figure 1j). Defence gene up-regulation in response to most peptides was stronger in V5 line. A stronger induction was triggered after elf18Ec and elf18Xcc compared to elf26Xfp treatment (Figure 1j), following the patterns observed for ROS production. Next, we evaluated whether the transgenic plants show enhanced resistance to CC and CVC. Detached leaves were infiltrated with the Xcc strain 306 bacterial suspension (104 CFU/mL) expressing GFP (Rigano et al., 2007). Canker lesions developed in all inoculated leaves 14 days after inoculation (dai), but with reduced severity in transgenic lines compared to the WT (Figure 1k). Notably, V5 only produced mild hyperplasic and water-soaked lesions, and petiole abscission, an advanced stage-mark of CC, was never observed (Figure 1k). Although V4 showed reduced symptom development, the bacterial population was not significantly different from the WT (Figure 1l). Pathogen growth in the V5 line was consistently reduced in the order of 3 log units (Figure 1l). In this transgenic line, bacterial spreading and growth were restrained, corroborating symptomatology results. To assess EFR-expressing citrus responses upon Xfp infection, a bacterial suspension (108 CFU/mL) of the 9a5c strain (Simpson et al., 2000) was petiole-inoculated on the first leaf of ten transgenic and WT plants. Disease severity and bacterial population assessed by qPCR were evaluated 18 months after inoculation at 5 and 30 cm above the inoculation point (aip). Seventy-one per cent of the plants showed colonization 5 cm aip, but not in more distal parts (Figure 1m). By contrast, WT and only two transgenic clones colonized distal parts at 30 cm aip (Figure 1m). Nevertheless, the symptom severity was much lower in transgenic plants compared to WT (Figure 1m). Transgenic lines without long-distance colonization were symptom-free during the extent of the evaluated time course (Figure 1m-n). These results indicate that the presence of EFR affects bacterial colonization and prevents systemic spread, a process associated with the release of X. fastidiosa OMVs (Ionescu et al., 2014). For more information on the methods, see https://doi.org/10.5281/zenodo.4723427. In summary, our results show that the expression of EFR in sweet orange confers ligand-dependent activation of defence responses, improving resistance against two citrus bacterial pathogens. With the aim to decrease or avoid the use of agrochemicals, genetic increase in crop resistance is economically viable and sustainable. It opens possibilities and encourages the use of pattern recognition receptors (PRRs) like EFR to confer broad-spectrum resistance as a strategic approach that may support biotechnology citrus breeding programmes. This is the first report of the successful transfer of EFR into a perennial crop, increasing resistance to X. fastidiosa, a devastating pathogen in citrus and olive groves across Europe and the Americas, for which no genetic or chemical methods are available. Our work describes a genetic strategy to improve Citrus resistance and potentially other perennials. We thank LFC Silva from IAC for greenhouse assistance. This work was supported by FAPESP (2013/10957-0), INCT-Citros (Fapesp-2014/50880-0; CNPq-465440/2014-2), and fellowships for LKM, DMM and NSTS (CAPES-001, FAPESP-2013/01412-0 and FAPESP-2019/01447-5). Work in the Zipfel laboratory is funded by the Gatsby Charitable Foundation and the 2Blades Foundation. Research in the Robatzek laboratory is funded by the Deutsche Forschungsgemeinschaft (DFG), grants RO-3550/14-1 and RO-3550/13-1. The authors declare no conflict of interest. AAS, SR and CZ designed this research. LKM, NSTS, DMM, RRSN and KR conducted experiments and analysed data. LKM and NSTS drafted the manuscript. LKM, NSTS, KR, SR, AAS and CZ contributed with intellectual input. AAS, SR and CZ provided analytical tools and revised the manuscript. All authors read and approved the final manuscript.